Nori Canine Hepcidin ELISA Kit Summary

Alternative names for Hepcidin: HAMP, HEPC

Alternative names for canine: Dog

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

Hepcidin is a protein that is encoded by the HAMP gene. Hepcidin is a key regulator of the entry of iron into the circulation in mammals.^[1] Hepcidin synthesis and secretion by the liver is controlled by iron stores within macrophages, inflammation (hepcidin is an acute phase reactant), hypoxia, and erythropoiesis. Macrophages communicate with the hepatocyte to regulate hepcidin release into the circulation via hemojuvelin, hereditary hemochromatosis protein, transferrin receptor 2, bone morphogenic protein 6 (BMP6), matriptase-2, neogenin, BMP receptors, and transferrin.^[2] Hepcidin is a regulator of iron metabolism. It inhibits iron transport by binding to the iron export channel ferroportin which is located in the basolateral plasma membrane of gut enterocytes and the plasma membrane of reticuloendothelial cells (macrophages), ultimately resulting in ferroportin breakdown in lysosomes. Inhibiting ferroportin prevents iron from being exported from the cell.^[3][4] In enterocytes, this prevents iron transmission into the hepatic portal system, thereby reducing dietary iron absorption. In macrophages, ferroportin inhibition causes iron sequestration within the cell. Increased hepcidin activity is partially responsible for reduced iron availability seen in anemia of chronic inflammation, such as kidney failure.^[5] Hepcidin has strong antimicrobial activity and is also active against the fungus Candida albicans, but has no activity against Pseudomonas aeruginosa. Erythroferrone may inhibit hepcidin and so providing more iron for hemoglobin synthesis in situations such as stress erythropoiesis.^[6] and vitamin D may decrease hepcidin.  During conditions in which the hepcidin level is abnormally high, such as inflammation, serum iron falls due to iron trapping within macrophages and liver cells and decreased gut iron absorption. This typically leads to anemia due to an inadequate amount of serum iron being available for developing red blood cells. When the hepcidin level is abnormally low such as in hemochromatosis, iron overload occurs due to increased ferroportin mediated iron efflux from storage and increased gut iron absorption.

References

1. Ganz T (2003). Blood. 102(3): 783–8.
2. Zhao N, et al. (2013). “Iron regulation by hepcidin”. J Clin Invest. 123(6): 2337–43.
3. Rossi E (2005). Clin Biochem Rev. 26(3): 47–9.
4. Gulec S, et al. (2014). Am J Physiol. GI Liver Physiol. 307(4): G397–409.
5. Ashby DR, et al. (2009). Kidney Int. 75(9): 976–81.
6. Kautz L, et al. (2014). Nature Genetics. 46(7): 678–84.